ABSTRACT Patients co-infected with HIV and tuberculosis (TB) exhibit accelerated progression of both diseases. A missed TB diagnosis in a HIV-positive patient, and vice versa, can have severe consequences, since mortality rates are very high in HIV/TB co-infected patients. The World Health Organization thus recommends HIV testing for TB patients and TB screening for HIV-affected patients. Early diagnosis of HIV/TB co-infections and appropriately timed interventions can improve health outcomes by slowing disease progression and reducing mortality and the spread of these infections. Given this urgent need for early diagnosis of HIV and TB, we propose to develop and validate a novel assay that rapidly and directly diagnoses both HIV and TB infections, using a sensitive nanoplasmon-enhanced scattering (nPES) platform to detect pathogen-specific (Ps) exosomes (PsExo) that carry TB- and/or HIV-derived antigens in patient serum samples. Ps antigens are often difficult to detect in circulation, due to low concentrations and potential masking by non-specific protein interactions or degradative enzymes, especially early in infection when antigen concentrations are low. Body fluids are rich in exosomes, and exosomes are rich sources of multiple Ps-antigens. Exosome enrichment effectively concentrates Ps- antigens and protects them from circulating hydrolases, thereby enhancing and improving detection. While use of PsExo as biomarkers for diagnosing specific infectious diseases is new and shows great promise, significant technical challenges, including the current lack of validated PxExo markers for specific diseases and simple and rapid assays for PsExo analysis, exist. We have recently demonstrated that we can use our nPES assay platform to quantify cancer-derived exosomes and diagnose early-stage pancreatic tumors from disease controls. We will adapt this approach to detect HIV and TB PsExo in this proposal. Aim 1 will address the first issue by identifying pathogenic proteins specifically or predominantly expressed on exosomes of infected cells and evaluating PsExo candidates by quantitative proteomics analysis of infected patients from Foundation for Innovative New Diagnostics (FIND) sample bank. Aim 2 will establish an optimized nPES array using Aim 1 PsExo biomarkers, apply machine learning to develop an nPES array that can effectively diagnose HIV, TB and HIV/TB patients based on nPES results, and validate these findings in an independent FIND cohort. This approach does not require a separate exosome isolation step and uses only microsample serum volumes. The proposed assay will benefit co-infected patients who are unable to produce sputum, extrapulmonary TB patients who often require invasive biopsies for diagnosis, and patients with acute HIV infections who are still HIV antibody negative.